This invention relates to modified plants. In particular, the invention relates to plants modified such that at least part of the plant (for example seeds of the plant) is capable of yielding a commercially useful oil.
Plants have long been a commercially valuable source of oil. Nutritional uses of plant-derived oils have hitherto been dominant, but attention is now turning additionally to plants as a source of industrially useful oils, for example as replacements for or improvements on mineral oils. Oil seeds, such as from rape, have a variety of lipids in them (Hildish and Williams, xe2x80x9cChemical Composition of Natural Lipidsxe2x80x9d, Chapman Hall, London, 1964). There is considerable interest in altering lipid composition by the use of recombinant DNA technology (e.g. Knauf, TIBtech, February 1987, 40-47), but by no means all of the goals have been realised to date for a variety of reasons, in spite of the ever-increasing sophistication of the technology.
Success in tailoring the lipid content of plant-derived oils requires a firm understanding of the biochemistry and genes involved. Broadly, two approaches are available. First, plants may be modified to permit the synthesis of fatty acids which are new (for the plant); so, for example, laurate and/or stearate may be synthesised in rape. Secondly, the pattern and/or extent of incorporation of fatty acids into the glycerol backbone of the lipid may be altered. It is with this latter approach that the present invention is concerned.
Lipids are formed in plants by the addition of fatty acid moieties onto the glycerol backbone by a series of acyl transferase enzymes. There are three positions on the glycerol molecule at which fatty acid (acyl) moieties may be substituted, and the substitution reached at each position is catalysed by a position-specific enzyme; the enzymes are glycerol-sn-3-phosphate acyltransferase (1-acyltransferase), 1-acyl-sn-glycerol-3-phosphate acyltransferase (2-acyltransferase) and sn-1,2-diacylglycerol acyltransferase (3-acyltransferase).
One, but not the only, current aim of xe2x80x9clipid engineeringxe2x80x9d in plants is to provide oils including lipids with a higher content of erucic (22:1) acid and/or oils containing trierucin. Erucic acid-containing lipids are commercially desirable for a number of purposes, particularly as replacements to or supplements for mineral oils in certain circumstances, as alluded to above. In the case of oil seed rape (Brassica napus), one of the most significant oil producing crops in cultivation today, the specificity of the 2-acyltransferase enzyme positively discriminates against the incorporation of erucic acid at position 2. So, even in those cultivars of rape which are able to incorporate erucic acid at positions 1 and 3, where there is no (or at least reduced) discrimination against erucic acid, only a maximum 66% of the fatty acids incorporated into triacyl glycerols can be erucic acid. Such varieties of rape are known as HEAR (high erucic acid rape) varieties.
It would therefore be desirable to produce plants, eg conventional oil seed rape as well as HEAR varieties, which contain useful levels of trierucin and/or contain higher levels of erucic acid and/or contain oils with erucic acid incorporated at position 2; the same can be said of oils of other vegetable oil crops such as maize, sunflower and soya, to name but a few examples. While in principle it may be thought possible to introduce into a desired plant DNA encoding a 2-acyltransferase of different fatty acid specificity, for example from a different plant, in practice there are a number of problems.
All enzymes involved in the acylation pathway for formation of triacylglycerols are membrane bound. These are the 1-acyltransferase, 2-acyltransferase and 3-acyltransferase which are present in the endoplasmic reticulum in the cytoplasm. They have not been purified. This makes working with them difficult and rules out the use of many conventional DNA cloning procedures. This difficulty does not, paradoxically, lie in the way of cloning the gene (or at least cDNA) encoding the Choroplastic 1-acyltransferase enzyme, which is soluble; in fact, recombinant DNA work has already been undertaken on this enzyme for a completely different purpose, namely the enhancement of chilling resistance in tobacco plant leaves, by Murata et al (Nature 356 710-713 (1992)).
Wolter et al, Fat Science Technology, 93, No 8: 288-89 (1991) suggested a strategy for cloning membrane bound enzymes such as 2-acyltransferases, although no exemplification was given.
WO-A-9413814 discloses a DNA sequence (and corresponding protein sequence) of a 2-acyltransferase. This sequence, which is derived from maize, is used to transform plants, such that the normal substrate specificity of the plants"" 2-acyltransferase is altered. This disclosure also included the use of a cDNA sequence for a 2-AT derived from maize to locate 2-ATs with a high degree of homology from both Brassica and Limnanthes species.
It has now been surprisingly found that there is in fact another 2-AT in Limnanthes which has no homologue in rape and which is seed specific. This 2-AT is able to incorporate erucic acid at the 2-position which the native 2-AT in rape, for example, is unable to do.
According to a first aspect of the invention, therefore, there is provided a recombinant or isolated DNA sequence, encoding an enzyme having membrane-bound 2-acyltransferase activity, and selected from:
(i) a DNA sequence comprising the DNA sequence of FIG. 3 (SEQ ID NO: 7) or its complementary strand,
(ii) nucleic acid sequences hybridising to the DNA sequence of FIG. 3 (SEQ ID NO: 7) or its complementary strand, under stringent conditions, and
(iii) nucleic acid sequences which would hybridise to the DNA sequence of FIG. 3 (SEQ ID NO: 7) or its complementary strand, but for the degeneracy of the genetic code.
Suitably, the DNA sequence of the invention comprises a DNA sequence as described in (i), (ii) or (iii) above which is the sequence of FIG. 3, or its complementary strand, or is one which has the characteristics of (ii) or (iii) where the sequence is the sequence of FIG. 3 (SEQ ID NO: 7)
Fragments of the above DNA sequences, for example of at least 15, 20, 30, 40 or 60 nucleotides in length, are also within the scope of the invention.
Suitable stringent conditions include salt solutions of approximately 0.9 molar at temperatures of from 35xc2x0 C. to 65xc2x0 C. More particularly, stringent hybridisation conditions include 6 x SSC, 5 x Denhardt""s solution, 0.5% SDS, 0.5% tetrasodium pyrophosphate and 50 xcexcg/ml denatured herring sperm DNA; washing may be for 2xc3x9730 minutes at 65xc2x0 C. in 1 x SSC, 0.1% SDS and 1xc3x9730 minutes in 0.2 x SSC, 0.1% SDS at 65xc2x0 C.
Recombinant DNA in accordance with the invention may be in the form of a vector, which may have sufficient regulatory sequences (such as a promoter) to direct gene expression. Vectors which are not expression vectors are useful for cloning purposes (as expression vectors themselves may be). Host cells (such as bacteria and plant cells) containing vectors in accordance with the invention themselves form part of the invention.
The 2-acyltransferase of the invention may be cloned directly, for example using complementation studies, from a DNA library of Limnanthes. For example, if E. coli is used as the complementation host, a mutant is chosen which is defective in the 2-acyltransferase; the DNA library from Limnanthes (e.g. L. douglasii) is transformed into the mutant complementation host; host cells containing the target acyltransferase gene can readily be selected using appropriate selective media and growth conditions. E. coli mutant JC201 is a suitable host for use in complementation studies relating to 2-acyltransferase.
Cloning the acyltransferase gene into a microbial host, such as a bacterium like E. coli, in such a way that the gene can be expressed has a particular advantage in that the substrate specificity of the acyltransferase gene can be assessed with membranes isolated from the microbial host before transformed plants are prepared, thereby saving considerably on research time. Such an assessment may be made by competitive substrate assays, in which differently detectably labelled candidate substrates for the enzyme compete with each other for incorporation into the glyceride. For example, 14C-erucyl CoA and 3H-oleoyl CoA can be used as competitive substrates for 2-acyltransferase, and the relative amounts of 14C or tritium uptake into glyceride can be measured. (As 2-acyltransferases have acceptor, glycerol-based, substrates and donor, fatty acid-based, substrates, the experiment can be carried out with different acceptors, such as 1-erucyl-glycerol-3-phosphate and 1-oleoyl-glycerol-3-phosphate.) A gene coding for an enzyme which donates erucic acid to the acceptor (particularly 1-erucyl-glycerol-3-phosphate) may by this means be identified as a DNA sequence of choice for further use in the invention as described below.
Suitably, the DNA sequence of the invention encodes an enzyme having membrane-bound 2-acyltransferase activity.
The DNA sequence of the invention can be used to produce proteins which they encode, if desired. Thus, in a second aspect, the present invention provides an isolated protein which is the expression product of a DNA sequence of the invention. The protein may be expressed by host cells harbouring DNA in the form of an expression vector. The protein, an enzyme having 2-acyltransferase activity, may have an amino acid sequence which is identical to or homologous with the sequence shown in FIG. 3 (SEQ ID NO: 4). The degree of homology will generally be greater than that of known proteins, and may be at least 40, 50, 60, 70, 80, 90, 95 or 99%. Suitably, the degree of homology will be 60% or greater, preferably 80% or greater and most preferably 90% or greater.
In a third aspect, the present invention provides an antibody capable of specifically binding to a protein of the invention.
In a fourth aspect of the invention, there is provided a plant having a 2-acyltransferase enzyme encoded by a DNA sequence as defined herein, wherein the enzyme is not a native enzyme of the plant.
While site-directed mutagenesis and/or other protein engineering techniques may be used to alter the specificity of an enzyme native to the plant, it is preferred that the plant be transgenic and incorporate an expressible 2-acyltransferase gene encoding the enzyme of the invention. For example, as described above, the 2-acyltransferase enzyme which does not discriminate against erucic acid, may be made by this means to express in a plant which would not normally incorporate erucic acid at the 2-position into triacylglycerides. An important embodiment of the invention relates to genetically engineered plants which contain trierucin. Such plants may thus also have higher levels of erucic acid incorporated into triacylglycerols than in corresponding non-engineered plants(eg. rape).
However, while a preferred approach is discussed above, the invention includes modified 2-acyltransferase proteins obtained by methods well known in the art. The essential feature that such proteins should possess is, of course, the specificity for incorporating erucic acid at position 2 of TAGs. However, using a variety of techniques modified enzymes can be obtained which have, for example, greater heat stability, improved kinetic characteristics or even improved specificity for erucic acid.
Suitable examples of such engineered plants include Brassica eg B. napus, B. campestris, B. Juncea or B. rapa, maize, sunflower or soya.
For the 2-acyltransferase transgene to be expressible, a promoter has to be operatively coupled to it. Because at the present state of the art it is difficult precisely to regulate the site of incorporation of a transgene into the host genome, it is preferred that the transgene be coupled to its promoter prior to transformation of the plant. Promoters useful in the invention may be temporal- and/or seed-specific, but there is no need for them to be so: constitutive promoters may also be used provided they are suitably strongly expressed in the seed and are easier to isolate. Other tissues are unlikely to be adversely affected if the transgene encoding the acyltransferase enzyme is expressed in them, as the availability of the fatty acid CoA substrates is effectively limited to the seed.
The promoter-transgene construct, once prepared, is introduced into plant cells by any suitable means. The invention extends to such plant cells. Preferably, DNA is transformed into plant cells using a disarmed Ti-plasmid vector and carried by Agrobacterium by procedures known in the art, for example as described in EP-A-0116718 and EP-A-0270822. Alternatively, the foreign DNA could be introduced directly into plant cells using an electrical discharge apparatus. This method is preferred where Agrobacterium is ineffective, for example where the recipient plant is monocotyledonous. Any other method that provides for the stable incorporation of the DNA within the nuclear DNA of any plant cell of any species would also be suitable. This includes species of plant which are not currently capable of genetic transformation.
The plants of the invention include ones which therefore have higher levels of erucic acid incorporated at the 2-position of their triacylglycerols (TAGs) as well as plants which contain trierucin.
Preferably DNA in accordance with the invention also contains a second chimeric gene (a xe2x80x9cmarkerxe2x80x9d gene) that enables a transformed plant or tissue culture containing the foreign DNA to be easily distinguished from other plants or tissue culture that do not contain the foreign DNA. Examples of such a marker gene include antibiotic resistance (Herrera-Estrella et al, EMBO J. 2(6) 987-95 (1983) and Herrera-Estrella et al, Nature 303 209-13 (1983)), herbicide resistance (EP-A-0242246) and glucuronidase (GUS) expression (EP-A-0344029). Expression of the marker gene is preferably controlled by a second promoter which allows expression in cells in culture, thus allowing selection of cells or tissue containing the marker at any stage of regeneration of the plant. The preferred second promoter is derived from the gene which encodes the 35S subunit of Cauliflower Mosaic Virus (CaMV) coat protein. However any other suitable second promoter could be used.
In one embodiment of the invention, the transgenic plant""s native 2-acyltransferase gene which corresponds to the transgene may be rendered at least partially inoperative or reduced in effectiveness by, for example, antisense or ribozyme techniques, as is known in the art.
A whole plant can be regenerated from a single transformed plant cell; and the invention therefore provides transgenic plants (or parts of them, such as propagating material) including DNA in accordance with the invention as described above. The regeneration can proceed by known methods.
Therefore, in a fifth aspect, the present invention provides a plant cell incorporating a DNA sequence of the invention.
In a sixth aspect, the invention provides seeds obtained from a plant of the invention.
By means of the invention, plants generating oil with a tailored lipid content may be produced. For example, plants which produce trierucin and/or incorporate erucic acid at position 2 of triacylglycerols (TAGs) can be engineered. In addition, the lipid composition of triacylglycerides in the plant may be substantially altered to produce triacylglycerides with a desired erucic acid content higher than has hitherto been possible. For example, oil seed rape (B. napus) may be transformed to produce oil whose triacylglyceride has an erucic acid content which is higher than that obtained in untransformed plants. Similarly for other oil producing crops.
Promoters which naturally drive 2-acyltransferases may also be obtained by hybridisation and/or restriction enzyme analysis and/or sequencing studies using the FIG. 3 (SEQ ID NO: 7) sequence.
In further aspects, the present invention provides:
(a) a method of generating oil, the method comprising cultivating a plant of the invention and harvesting oil produced by the plant or a part (particularly seeds) thereof;
(b) oil obtained from a plant of the invention, or a part thereof, or from seeds of the invention which has erucic acid incorporated at the 2-position of its TAGs;
(c) oil obtained from a plant of the invention, or a part thereof, or from seeds of the invention which contains trierucin;
(d) a microbial host transformed with a DNA sequence of the invention;
(e) an oil seed rape plant, or other oil producing crop plant, containing trierucin;
(f) an oil seed rape plant, or other oil producing crop plant, having erucic acid incorporated at the 2-position of its TAGs; and
(g) a transgenic plant which expresses in at least some of its cells a DNA sequence of the invention. In particular, the DNA sequence is expressed in the seeds of the plant.
Preferred features of each aspect of the invention are as for each other aspect mutatis mutandis.